Sialylated Glycoproteins

ABSTRACT

Pharmaceutical preparations containing polypeptides having particular sialylation patterns, and methods for the treatment of immune-related thrombocytopenia with such preparations, are described.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/985,288, filed on May 21, 2018, which is a continuation of U.S.patent application Ser. No. 15/028,917, filed on Apr. 12, 2016 (nowabandoned), which is national stage application under 35 U.S.C. § 371 ofInternational Application Number PCT/US2014/060363, filed on Oct. 14,2014, which claims the benefit of U.S. Provisional Application No.61/891,778, filed Oct. 16, 2013, which are hereby incorporated byreference in their entirety.

BACKGROUND

Therapeutic glycoproteins are an important class of therapeuticbiotechnology products, and therapeutic Fc containing glycoproteins,such as IVIg, Fc-receptor fusions, and antibodies (including murine,chimeric, humanized, and human antibodies and fragments thereof) accountfor the majority of therapeutic biologic products.

SUMMARY OF THE INVENTION

The invention encompasses, in part, the discovery that Fc-containingpolypeptides that include branched glycans and that are di-sialylated onthe branched glycan (e.g., on an α 1,3 and/or a 1,6 arm in the Fcregion's N-linked glycosylation site), with, e.g., a NeuAc-α 2,6-Galterminal linkage, exhibit improved biological activity, e.g., relativeto a reference glycoprotein, e.g., in the treatment of hematologicaldisease, e.g., immune-related thrombocytopenia (ITP). The presentdisclosure provides, in part, methods for treating hematologicaldisease, e.g., immune-related thrombocytopenia and related diseases byadministering compositions containing such Fc-containing polypeptides aswell as methods for evaluating, identifying, and/or producing (e.g.,manufacturing) such polypeptides.

In one aspect, the invention features a pharmaceutical preparationformulated for subcutaneous administration (e.g., at a concentration of50-250 mg/mL, e.g., 50-100 mg/mL, 75-125 mg/mL, 100-150 mg/mL, 125-175mg/mL, 150-200 mg/mL, 175-225 mg/mL, 200-250 mg/mL). This preparationincludes polypeptides having an Fc region, wherein at least 50% (e.g.,60%, 70%, 80%, 82%, 85%, 87%, 90%, 92%, 94%, 95%, 97%, 98% up to andincluding 100%) of branched glycans on the Fc region are di-sialylatedby way of NeuAc-α 2,6-Gal terminal linkages. In some embodiments, lessthan 50% (e.g., less than 40%, 30%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%)of branched glycans on the Fc region are mono-sialylated (e.g., on the α1,3 arm or the α 1,6 arm) by way of a NeuAc-α 2,6-Gal terminal linkage.

In another aspect, the invention features a pharmaceutical preparationincluding polypeptides having an Fc region, wherein at least 50% (e.g.,60%, 70%, 80%, 82%, 85%, 87%, 90%, 92%, 94%, 95%, 97%, 98% up to andincluding 100%) of branched glycans on the Fc region are di-sialylatedby way of NeuAc-α 2,6-Gal terminal linkages and less than 50% (e.g.,less than 40%, 30%, 20%, 10%, 15%, 5%, 4%, 3%, 2%, 1%) of branchedglycans on the Fc region are mono-sialylated on the α 1,3 arm by way ofa NeuAc-α 2,6-Gal terminal linkage.

In another aspect, the invention features a pharmaceutical preparationcomprising polypeptides having an Fc region, wherein at least 50% (e.g.,60%, 70%, 80%, 82%, 85%, 87%, 90%, 92%, 94%, 95%, 97%, 98% up to andincluding 100%) of branched glycans on the Fc region are di-sialylatedby way of NeuAc-α 2,6-Gal terminal linkages and less than 50% (e.g.,less than 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%) of branched glycans onthe Fc region are mono-sialylated on the α 1,6 arm by way of a NeuAc-α2,6-Gal terminal linkage.

In another aspect, the invention features a pharmaceutical preparationcomprising polypeptides having an Fc region, wherein at least 85% ofbranched glycans on the Fc region are di-sialylated by way of NeuAc-α2,6-Gal terminal linkages.

In some embodiments of any of the foregoing preparations, thepolypeptides consist essentially of an Fc region. In other embodimentsof any of the foregoing preparations, the polypeptides further include aFab region, a heterologous polypeptide sequence such as a biologicalreceptor sequence (e.g., the polypeptides are Fc-receptor fusionproteins), or a heterologous non-polypeptide moiety.

In certain embodiments, at least 10% (e.g., 20%, 30%, 40%, 50%, 60% 70%or more) of branched glycans on the Fab region or heterologouspolypeptide sequence of the polypeptides are mono-sialylated ordi-sialylated. In other embodiments, less than 80% (e.g., 70%, 60, 50%,40%, 30%, 20%, 10%, 5% or less) of branched glycans on the Fab region orheterologous polypeptide sequence of the polypeptides aremono-sialylated or di-sialylated.

In some embodiments of any of the foregoing preparations, thepolypeptides are recombinant polypeptides. In other embodiments of anyof the foregoing preparations, the polypeptides are derived from plasma,e.g., human plasma. In certain embodiments, the polypeptides are IgGpolypeptides (e.g., IgG1, IgG2, IgG3 or IgG4) or the polypeptidesconsist essentially of an Fc region derived from IgG polypeptides.

In another aspect, the invention features a method of increasingreticulated platelets in a subject in need thereof, comprisingadministering to the subject any one of the foregoing preparations.

In another aspect, the invention features a method of producing newplatelets in a subject in need thereof, comprising administering to thesubject any one of the foregoing preparations.

In another aspect, the invention features a method of increasingreticulated platelets or producing new platelets in a subject in needthereof, comprising administering to the subject a pharmaceuticalpreparation comprising polypeptides comprising an Fc region, wherein atleast 85% of branched glycans on the Fc region are di-sialylated by wayof NeuAc-α 2,6-Gal terminal linkages.

In some embodiments of any of the foregoing methods, the subject is notbeing treated with thrombopoietin or a thrombopoietin receptor agonist(e.g., romiplostim, eltrombopag). In some embodiments of any of theforegoing methods, the subject has failed treatment with thrombopoietinor a thrombopoietin receptor agonist (e.g., romiplostim, eltrombopag).In other embodiments of any of the foregoing methods, the subject has ahematological disease such as immune-related thrombocytopenia. Incertain embodiments of any of the foregoing methods, the method furtherincludes, after the administering step, the step of determining thetotal platelet count and/or the reticulated platelet count in thesubject, e.g., wherein the total platelet count and/or the reticulatedplatelet count increases as a result of the administering step. In someembodiments, the method further includes after the determining step, thestep of adjusting the dose of the administered pharmaceuticalpreparation.

DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic illustration of a common core pentasaccharide(Man)₃(GlcNAc)(GlcNAc) of N-glycans.

FIG. 2 is a schematic illustration of an IgG antibody molecule.

FIG. 3A depicts an exemplary ST6 sialyltransferase amino acid sequence(SEQ ID NO:1). FIG. 3B depicts an exemplary ST6 sialyltransferase aminoacid sequence (SEQ ID NO:2). FIG. 3C depicts an exemplary ST6sialyltransferase amino acid sequence (SEQ ID NO:3).

FIG. 4 is a schematic illustration of a reaction scheme for ST6sialyltransferase (fucose: triangles, N-acetylglucosamine: squares,mannose: dark circles, galactose: light circles, sialic acid: diamonds).

FIG. 5 is a graphic representation of relative abundance of glycans atvarious times during a sialylation reaction with ST6 sialyltransferase.

DETAILED DESCRIPTION OF THE INVENTION

Antibodies are glycosylated at conserved positions in the constantregions of their heavy chain. For example, IgG antibodies have a singleN-linked glycosylation site at Asn297 of the CH2 domain. Each antibodyisotype has a distinct variety of N-linked carbohydrate structures inthe constant regions. For human IgG, the core oligosaccharide normallyconsists of GlcNAc₂Man₃GlcNAc, with differing numbers of outer residues.Variation among individual IgG's can occur via attachment of galactoseand/or galactose-sialic acid at one or both terminal GlcNAc or viaattachment of a third GlcNAc arm (bisecting GlcNAc).

The present disclosure encompasses, in part, pharmaceutical preparationsincluding polypeptides having an Fc region having particular levels ofbranched glycans that are sialylated on both of the branched glycans inthe Fc region (e.g., with a NeuAc-α 2,6-Gal terminal linkage). Thelevels can be measured on an individual Fc region (e.g., the number ofbranched glycans that are sialylated on an α1,3 arm, an α1,6 arm, orboth, of the branched glycans in the Fc region), or on the overallcomposition of a preparation of polypeptides (e.g., the number orpercentage of branched glycans that are sialylated on an α1,3 arm, anα1,6 arm, or both, of the branched glycans in the Fc region in apreparation of polypeptides).

The inventors have discovered that Fc-region containing polypeptideshaving branched glycans that are preferentially di-sialylated (e.g.,with NeuAc-α 2,6-Gal terminal linkages) exhibit improved biologicalactivity, e.g., relative to a reference glycoprotein, and are useful inthe treatment of immune-related thrombocytopenia and related diseases.

Preparations useful herein can be obtained from any source. In someinstances, providing or obtaining a preparation (e.g., such as abiologic drug substance or a precursor thereof), e.g., that is orincludes a polypeptide, can include providing a host cell, e.g., amammalian host cell (e.g., a CHO cell) that is genetically engineered toexpress a polypeptide (e.g., a genetically engineered cell); culturingthe host cell under conditions suitable to express the polypeptide(e.g., mRNA and/or protein); and, optionally, purifying the expressedpolypeptide, e.g., in the form of a recombinant fusion protein) from thecultured cell, thereby producing a preparation.

Definitions

As used herein, “acquire or acquiring (e.g., acquiring information)”means obtaining possession of a physical entity, or a value, e.g., anumerical value, by “directly acquiring” or “indirectly acquiring” thephysical entity or value. “Directly acquiring” means performing aprocess (e.g., performing an assay or test on a sample or “analyzing asample” as that term is defined herein) to obtain the physical entity orvalue. “Indirectly acquiring” refers to receiving the physical entity orvalue from another party or source (e.g., a third party laboratory thatdirectly acquired the physical entity or value). “Directly acquiring” aphysical entity includes performing a process, e.g., analyzing a sample,that includes a physical change in a physical substance, e.g., astarting material. Exemplary changes include making a physical entityfrom two or more starting materials, shearing or fragmenting asubstance, separating or purifying a substance, combining two or moreseparate entities into a mixture, performing a chemical reaction thatincludes breaking or forming a covalent or non-covalent bond. “Directlyacquiring” a value includes performing a process that includes aphysical change in a sample or another substance, e.g., performing ananalytical process which includes a physical change in a substance,e.g., a sample, analyte, or reagent (sometimes referred to herein as“physical analysis”), performing an analytical method, e.g., a methodwhich includes one or more of the following: separating or purifying asubstance, e.g., an analyte, or a fragment or other derivative thereof,from another substance; combining an analyte, or fragment or otherderivative thereof, with another substance, e.g., a buffer, solvent, orreactant; or changing the structure of an analyte, or a fragment orother derivative thereof, e.g., by breaking or forming a covalent ornon-covalent bond, between a first and a second atom of the analyte; orby changing the structure of a reagent, or a fragment or otherderivative thereof, e.g., by breaking or forming a covalent ornon-covalent bond, between a first and a second atom of the reagent.Exemplary analytical methods are shown in Table 1.

As used herein, the term “antibody” refers to a polypeptide thatincludes at least one immunoglobulin variable region, e.g., an aminoacid sequence that provides an immunoglobulin variable domain orimmunoglobulin variable domain sequence. For example, an antibody caninclude a heavy (H) chain variable region (abbreviated herein as V_(H)),and a light (L) chain variable region (abbreviated herein as V_(L)). Inanother example, an antibody includes two heavy (H) chain variableregions and two light (L) chain variable regions. The term “antibody”encompasses antigen-binding fragments of antibodies (e.g., single chainantibodies, Fab, F(ab′)₂, Fd, Fv, and dAb fragments) as well as completeantibodies, e.g., intact immunoglobulins of types IgA, IgG, IgE, IgD,IgM (as well as subtypes thereof). The light chains of theimmunoglobulin can be of types kappa or lambda.

As used herein, a “batch” of a preparation refers to a single productionrun. Evaluation of different batches thus means evaluation of differentproduction runs or batches. As used herein “sample(s)” refer toseparately procured samples. For example, evaluation of separate samplescould mean evaluation of different containers or vials of the same batchor from different batches. A batch can include a drug substance batch ora drug product batch.

As used herein, the term “constant region” refers to a polypeptide thatcorresponds to, or is derived from, one or more constant regionimmunoglobulin domains of an antibody. A constant region can include anyor all of the following immunoglobulin domains: a C_(H)1 domain, a hingeregion, a C_(H)2 domain, a C_(H)3 domain (derived from an IgA, IgD, IgG,IgE, or IgM), and a C_(H)4 domain (derived from an IgE or IgM).

As used herein, “evaluating,” e.g., in the evaluation/evaluating,identifying, and/or producing aspects disclosed herein, means reviewing,considering, determining, assessing, analyzing, measuring, and/ordetecting the presence, absence, level, and/or ratio of one or moreparameters in a preparation to provide information pertaining to the oneor more parameters. In some instances, evaluating can include performinga process that involves a physical change in a sample or anothersubstance, e.g., a starting material. Exemplary changes include making aphysical entity from two or more starting materials, shearing orfragmenting a substance, separating or purifying a substance, combiningtwo or more separate entities into a mixture, performing a chemicalreaction that includes breaking or forming a covalent or non-covalentbond. “Evaluating” can include performing an analytical process whichincludes a physical change in a substance, e.g., a sample, analyte, orreagent (sometimes referred to herein as “physical analysis”),performing an analytical method, e.g., a method which includes one ormore of the following: separating or purifying a substance, e.g., ananalyte, or a fragment or other derivative thereof, from anothersubstance; combining an analyte, or fragment or other derivativethereof, with another substance, e.g., a buffer, solvent, or reactant;or changing the structure of an analyte, or a fragment or otherderivative thereof, e.g., by breaking or forming a covalent ornon-covalent bond, between a first and a second atom of the analyte; orby changing the structure of a reagent, or a fragment or otherderivative thereof, e.g., by breaking or forming a covalent ornon-covalent bond, between a first and a second atom of the reagent.

As used herein, the term “Fc region” refers to a dimer of two “Fcpolypeptides,” each “Fc polypeptide” including the constant region of anantibody excluding the first constant region immunoglobulin domain. Insome embodiments, an “Fc region” includes two Fc polypeptides linked byone or more disulfide bonds, chemical linkers, or peptide linkers. “Fcpolypeptide” refers to the last two constant region immunoglobulindomains of IgA, IgD, and IgG, and the last three constant regionimmunoglobulin domains of IgE and IgM, and may also include part or theentire flexible hinge N-terminal to these domains. For IgG, “Fcpolypeptide” comprises immunoglobulin domains Cgamma2 (Cγ2) and Cgamma3(Cγ3) and the lower part of the hinge between Cgamma1 (Cγ1) and Cγ2.Although the boundaries of the Fc polypeptide may vary, the human IgGheavy chain Fc polypeptide is usually defined to comprise residuesstarting at T223 or C226 or P230, to its carboxyl-terminus, wherein thenumbering is according to the EU index as in Kabat et al. (1991, NIHPublication 91-3242, National Technical Information Services,Springfield, Va.). For IgA, Fc polypeptide comprises immunoglobulindomains Calpha2 (Cα2) and Calpha3 (Cα3) and the lower part of the hingebetween Calpha1 (Cα1) and Cα2. An Fc region can be synthetic,recombinant, or generated from natural sources such as IVIg.

An “Fc region-containing polypeptide” is a polypeptide that includes allor a substantial portion of an Fc region. Examples of an Fcregion-containing polypeptide preparation include, e.g., a preparationof Fc fragments, a preparation of antibody molecules, a preparation ofFc-fusion proteins (e.g., an Fc-receptor fusion protein), and apreparation of pooled, polyvalent immunoglobulin molecules (e.g., IVIg).Such an Fc region-containing polypeptide may be recombinant (e.g., arecombinant Fc fragment preparation or a recombinant antibodypreparation) or naturally derived (such as IVIg).

As used herein, “glycan” is a sugar, which can be monomers or polymersof sugar residues, such as at least three sugars, and can be linear orbranched. A “glycan” can include natural sugar residues (e.g., glucose,N-acetylglucosamine, N-acetyl neuraminic acid, galactose, mannose,fucose, hexose, arabinose, ribose, xylose, etc.) and/or modified sugars(e.g., 2′-fluororibose, 2′-deoxyribose, phosphomannose, 6′sulfoN-acetylglucosamine, etc.). The term “glycan” includes homo andheteropolymers of sugar residues. The term “glycan” also encompasses aglycan component of a glycoconjugate (e.g., of a polypeptide,glycolipid, proteoglycan, etc.). The term also encompasses free glycans,including glycans that have been cleaved or otherwise released from aglycoconjugate.

As used herein, the term “glycoprotein” refers to a protein thatcontains a peptide backbone covalently linked to one or more sugarmoieties (i.e., glycans). The sugar moiety(ies) may be in the form ofmonosaccharides, disaccharides, oligosaccharides, and/orpolysaccharides. The sugar moiety(ies) may comprise a single unbranchedchain of sugar residues or may comprise one or more branched chains.Glycoproteins can contain O-linked sugar moieties and/or N-linked sugarmoieties.

As used herein, “immune-related thrombocytopenia” refers to disorders inwhich there is a relative decrease of platelets in the blood caused byincreased destruction of platelets by the immune system. Non-limitingexamples of immune-related thrombocytopenia disorders include idiopathicthrombocytopenic purapura, neonatal alloimmune thrombocytopenia,post-transfusion purapura, and systemic lupus erythematosus relatedthrombocytopenia.

As used herein, “IVIg” is a preparation of pooled, polyvalent IgG,including all four IgG subgroups, extracted from plasma of at least1,000 human donors. IVIg is approved as a plasma protein replacementtherapy for immune deficient patients. The level of IVIg Fc glycansialylation varies between about 10-20% among IVIg preparations. As usedherein, the term “derived from IVIg” refers to polypeptides which resultfrom manipulation of IVIg. For example, polypeptides purified from IVIg(e.g., enriched for sialylated IgGs, modified IVIg (e.g., IVIg IgGsenzymatically sialylated), or Fc regions of IVIg (e.g., papain digestedand sialylated) are derived from IVIg.

As used herein, an “N-glycosylation site of an Fc polypeptide” refers toan amino acid residue within an Fc polypeptide to which a glycan isN-linked. In some embodiments, an Fc region contains a dimer of Fcpolypeptides, and the Fc region comprises two N-glycosylation sites, oneon each Fc polypeptide.

As used herein “percent (%) of branched glycans” refers to the number ofmoles of glycan X relative to total moles of glycans present, wherein Xrepresents the glycan of interest.

As used herein “percent (%) sequence identity” with respect to asequence is defined as the percentage of amino acid residues ornucleotides in a candidate sequence that are identical with the aminoacid residues or nucleotides in the reference sequence, after aligningthe sequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity. Gaps can be introduced in one or both of afirst and a second amino acid or nucleic acid sequence for optimalalignment and non-homologous sequences can be disregarded for comparisonpurposes. Alignment for purposes of determining percent sequenceidentity can be achieved in various ways that are within the skill inthe art, for instance, using publicly available computer software suchas BLAST, ALIGN or Megalign (DNASTAR) software. Those skilled in the artcan determine appropriate parameters for measuring alignment, includingany algorithms needed to achieve maximal alignment over the full lengthof the sequences being compared. In one embodiment, the length of areference sequence aligned for comparison purposes is at least 30%,e.g., at least 40%, e.g., at least 50%, 60%, 70%, 80%, 90%, or 100% ofthe length of the reference sequence. The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. In some instances a product will includeamino acid variants, e.g., species that differ at terminal residues,e.g., at one, two, three, or four N-terminal residues and/or oneC-terminal residue. In instances of such cases the sequence identitywhich is compared is the identity between the primary amino acidsequences of the most abundant active species in each of the productsbeing compared. In some instances sequence identity refers to the aminoacid sequence encoded by a nucleic acid that can be used to make theproduct.

The term “pharmaceutically effective amount” or “therapeuticallyeffective amount” refers to an amount (e.g., dose) effective in treatinga patient, having a disorder or condition described herein. It is alsoto be understood herein that a “pharmaceutically effective amount” maybe interpreted as an amount giving a desired therapeutic effect, eithertaken in one dose or in any dosage or route, taken alone or incombination with other therapeutic agents.

“Pharmaceutical preparations” and “pharmaceutical products” can beincluded in kits containing the preparation or product and instructionsfor use.

“Pharmaceutical preparations” and “pharmaceutical products” generallyrefer to compositions in which the final predetermined level ofsialylation has been achieved, and which are free of process impurities.To that end, “pharmaceutical preparations” and “pharmaceutical products”are substantially free of ST6Gal sialyltransferase and/or sialic aciddonor (e.g., cytidine 5′-monophospho-N-acetyl neuraminic acid) or thebyproducts thereof (e.g., cytidine 5′-monophosphate).

“Pharmaceutical preparations” and “pharmaceutical products” aregenerally substantially free of other components of a cell in which theglycoproteins were produced (e.g., the endoplasmic reticulum orcytoplasmic proteins and RNA), if recombinant.

As used herein, “polynucleotide” (or “nucleotide sequence” or “nucleicacid molecule”) refers to an oligonucleotide, nucleotide, orpolynucleotide, and fragments or portions thereof, and to DNA or RNA ofgenomic or synthetic origin, which may be single- or double-stranded,and represent the sense or anti-sense strand.

As used herein, “polypeptide” (or “amino acid sequence” or “protein”)refers to a glycoprotein, oligopeptide, peptide, polypeptide, or proteinsequence, and fragments or portions thereof, and to naturally occurringor synthetic molecules. “Amino acid sequence” and like terms, such as“polypeptide” or “protein,” are not meant to limit the indicated aminoacid sequence to the complete, native amino acid sequence associatedwith the recited protein molecule.

“Predetermined level” as used herein, refers to a pre-specifiedparticular level of one or more particular glycans, e.g., branchedglycans having a sialic acid on an α1,3 arm, and/or branched glycanshaving a sialic acid on an α1,6 arm, and/or branched glycans having asialic acid on an α1,3 arm and on an α1,6 arm. In some embodiments, apredetermined level is an absolute value or range. In some embodiments,a predetermined level is a relative value. In some embodiments, apredetermined level is the same as or different (e.g., higher or lowerthan) a level of one or more particular glycans (e.g., branched glycanshaving a sialic acid on an α1,3 arm, and/or branched glycans having asialic acid on an α1,6 arm, and/or branched glycans having a sialic acidon an α1,3 arm and on an α1,6 arm) in a reference, e.g., a referencepolypeptide product, or a level specified in a reference document suchas a pharmaceutical specification, a monograph, alert limit, or masterbatch record for a pharmaceutical product.

In some embodiments, a predetermined level is an absolute level or rangeof (e.g., number of moles of) one or more glycans (e.g., branchedglycans having a sialic acid on an α1,3 arm, and/or branched glycanshaving a sialic acid on an α1,6 arm, and/or branched glycans having asialic acid on an α1,3 arm and on an α1,6 arm) in a polypeptidepreparation. In some embodiments, a predetermined level is a level orrange of one or more glycans (e.g., branched glycans having a sialicacid on an α1,3 arm, and/or branched glycans having a sialic acid on anα1,6 arm, and/or branched glycans having a sialic acid on an α1,3 armand on an α1,6 arm) in a polypeptide preparation relative to total levelof glycans in the polypeptide preparation. In some embodiments, apredetermined level is a level or range of one or more glycans (e.g.,branched glycans having a sialic acid on an α1,3 arm, and/or branchedglycans having a sialic acid on an α1,6 arm, and/or branched glycanshaving a sialic acid on an α1,3 arm and on an α1,6 arm) in a polypeptidepreparation relative to total level of sialylated glycans in thepolypeptide preparation. In some embodiments, a predetermined level isexpressed as a percent.

By “purified” (or “isolated”) refers to a polynucleotide or apolypeptide that is removed or separated from other components presentin its natural environment. For example, an isolated polypeptide is onethat is separated from other components of a cell in which it wasproduced (e.g., the endoplasmic reticulum or cytoplasmic proteins andRNA). An isolated polynucleotide is one that is separated from othernuclear components (e.g., histones) and/or from upstream or downstreamnucleic acids. An isolated polynucleotide or polypeptide can be at least60% free, or at least 75% free, or at least 90% free, or at least 95%free from other components present in natural environment of theindicated polynucleotide or polypeptide.

“Reference polypeptide” refers to a polypeptide having substantially thesame amino acid sequence as (e.g., having about 95-100% identical aminoacids of) a polypeptide described herein, e.g., a polypeptide to whichit is compared. In some embodiments, a reference polypeptide is atherapeutic polypeptide described herein, e.g., an FDA approvedtherapeutic polypeptide.

As used herein, the term “sialylated” refers to a glycan having aterminal sialic acid. The term “mono-sialylated” refers to branchedglycans having one terminal sialic acid, e.g., on an α1,3 arm or an α1,6arm. The term “di-sialylated” refers to a branched glycan having aterminal sialic acid on two arms, e.g., both an α1,3 arm and an α1,6arm.

As used herein, the term “ST6 sialyltransferase” refers to a polypeptidewhose amino acid sequence includes at least one characteristic sequenceof and/or shows at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%,91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%,77%, 76%, 75%, 74%, 73%, 72%, 71% or 70% identity with a proteininvolved in transfer of a sialic acid to a terminal galactose of aglycan through an α2,6 linkage (e.g., ST6 Gal-I). A wide variety of ST6sialyltransferase sequences are known in the art, such as thosedescribed herein; in some embodiments, an ST6 sialyltransferase sharesat least one characteristic sequence of and/or shows the specifieddegree of overall sequence identity with one of the ST6sialyltransferases set forth herein (each of which may be considered a“reference” ST6 sialyltransferase). In some embodiments, an ST6sialyltransferase as described herein shares at least one biologicalactivity with a reference ST6 sialyltransferase as set forth herein. Insome such embodiment, the shared biological activity relates to transferof a sialic acid to a glycan.

The term “subject,” as used herein, means any subject for whomdiagnosis, prognosis, or therapy is desired. In one embodiment, thesubject is a human.

The term “thrombopoietin receptor agonist,” as used herein, refers topharmaceutical agents that stimulate platelet production in the bonemarrow through interaction with the thrombopoietin receptor.

The term “treatment” or “treating,” as used herein, refers toadministering a therapy in an amount, manner, and/or mode effective toimprove a condition, symptom, or parameter associated with a disorder orcondition or to prevent or reduce progression of a disorder or conditionto a degree detectable to one skilled in the art. An effective amount,manner, or mode can vary depending on the subject and may be tailored tothe subject. The term “not being treated,” as used herein, means asubject is not currently being administered a therapy.

As used herein, the terms “coupled,” “linked,” “joined,” “fused,” and“fusion” are used interchangeably. These terms refer to the joiningtogether of two more elements or components by whatever means, includingchemical conjugation or recombinant means.

While the present disclosure provides exemplary units and methods forthe evaluation, identification, and production methods disclosed herein,a person of ordinary skill in the art will appreciate that performanceof the evaluation, identification, and production methods herein is notlimited to use of those units and/or methods. For example, “percent ofbranched glycans” provided herein are generally described, as a valuefor a glycan or structure relative to total glycan or structure on amol/mol basis. A person of skill in the art understands that althoughthe use of other metrics or units (e.g., mass/mass, mole percent vs.weight percent) to measure a described parameter might give rise todifferent absolute values than those described herein, a testpreparation meets a disclosed target value even if other units ormetrics are used, as long as the test preparation meets the hereindisclosed value when the herein disclosed units and metrics are used,e.g., allowing for the sensitivity (e.g., analytical variability) of themethod being used to measure the value.

I. Polypeptides

Examples of an Fc region-containing polypeptide preparation include,e.g., a preparation of Fc fragments, a preparation of antibodymolecules, a preparation of Fc-fusion proteins (e.g., an Fc-receptorfusion protein), and a preparation of pooled, polyvalent immunoglobulinmolecules (e.g., IVIg). Fc region-containing polypeptides may berecombinant or naturally derived.

Naturally derived polypeptides that can be used in the methods of theinvention include, for example, intravenous immunoglobulin (IVIg) andpolypeptides derived from IVIg (e.g., polypeptides purified from IVIg(e.g., enriched for sialylated IgGs), modified IVIg (e.g., IVIg IgGsenzymatically sialylated), or Fc regions of IVIg (e.g., papain digestedand sialylated)).

Recombinant Fc region-containing polypeptides that can be used in themethods of the invention can be, for example, expressed in and purifiedfrom CHO cells and sialylated using human ST6-Gal sialtransferase enzyme(expressed in and purified from E. coli cells) or expressed in andpurified from CHO cells and sialylated using human ST6-Galsialtransferase enzyme (expressed in and purified from CHO cells).

A. N-Linked Glycosylation

N-linked oligosaccharide chains are added to a protein in the lumen ofthe endoplasmic reticulum. Specifically, an initial oligosaccharide(typically 14-sugar) is added to the amino group on the side chain of anasparagine residue contained within the target consensus sequence ofAsn-X-Ser/Thr, where X may be any amino acid except proline. Thestructure of this initial oligosaccharide is common to most eukaryotes,and contains three glucose, nine mannose, and two N-acetylglucosamineresidues. This initial oligosaccharide chain can be trimmed by specificglycosidase enzymes in the endoplasmic reticulum, resulting in a short,branched core oligosaccharide composed of two N-acetylglucosamine andthree mannose residues. One of the branches is referred to in the art asthe “α 1,3 arm,” and the second branch is referred to as the “α 1,6arm,” as denoted in FIG. 1.

N-glycans can be subdivided into three distinct groups called “highmannose type,” “hybrid type,” and “complex type,” with a commonpentasaccharide core (Man (α 1,6)-(Man(α 1,3))-Man(β 1,4)-GlcpNAc(β1,4)-GlcpNAc(β 1,N)-Asn) occurring in all three groups.

After initial processing in the endoplasmic reticulum, the polypeptideis transported to the Golgi where further processing may take place. Ifthe glycan is transferred to the Golgi before it is completely trimmedto the core pentasaccharide structure, it results in a “high-mannoseglycan.”

Additionally or alternatively, one or more monosaccharides units ofN-acetylglucosamine may be added to the core mannose subunits to form a“complex glycan.” Galactose may be added to the N-acetylglucosaminesubunits, and sialic acid subunits may be added to the galactosesubunits, resulting in chains that terminate with any of a sialic acid,a galactose or an N-acetylglucosamine residue. Additionally, a fucoseresidue may be added to an N-acetylglucosamine residue of the coreoligosaccharide. Each of these additions is catalyzed by specificglycosyl transferases.

“Hybrid glycans” comprise characteristics of both high-mannose andcomplex glycans. For example, one branch of a hybrid glycan may compriseprimarily or exclusively mannose residues, while another branch maycomprise N-acetylglucosamine, sialic acid, galactose, and/or fucosesugars.

Sialic acids are a family of 9-carbon monosaccharides with heterocyclicring structures. They bear a negative charge via a carboxylic acid groupattached to the ring as well as other chemical decorations includingN-acetyl and N-glycolyl groups. The two main types of sialyl residuesfound in polypeptides produced in mammalian expression systems areN-acetyl-neuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc).These usually occur as terminal structures attached to galactose (Gal)residues at the non-reducing termini of both N- and O-linked glycans.The glycosidic linkage configurations for these sialyl groups can beeither a 2,3 or a 2,6.

Fc regions are are glycosylated at conserved, N-linked glycosylationsites. For example, each heavy chain of an IgG antibody has a singleN-linked glycosylation site at Asn297 of the C_(H)2 domain. IgAantibodies have N-linked glycosylation sites within the C_(H)2 andC_(H)3 domains, IgE antibodies have N-linked glycosylation sites withinthe C_(H)3 domain, and IgM antibodies have N-linked glycosylation siteswithin the C_(H)1, C_(H)2, C_(H)3, and C_(H)4 domains.

Each antibody isotype has a distinct variety of N-linked carbohydratestructures in the constant regions. For example, IgG has a singleN-linked biantennary carbohydrate at Asn297 of the C_(H)2 domain in eachFc polypeptide of the Fc region, which also contains the binding sitesfor C1q and FcγR. For human IgG, the core oligosaccharide normallyconsists of GlcNAc₂Man₃GlcNAc, with differing numbers of outer residues.Variation among individual IgG can occur via attachment of galactoseand/or galactose-sialic acid at one or both terminal GlcNAc or viaattachment of a third GlcNAc arm (bisecting GlcNAc).

B. Antibodies

The basic structure of an IgG antibody is illustrated in FIG. 2. Asshown in FIG. 2, an IgG antibody consists of two identical lightpolypeptide chains and two identical heavy polypeptide chains linkedtogether by disulphide bonds. The first domain located at the aminoterminus of each chain is variable in amino acid sequence, providing theantibody binding specificities found in each individual antibody. Theseare known as variable heavy (V_(H)) and variable light (V_(L)) regions.The other domains of each chain are relatively invariant in amino acidsequence and are known as constant heavy (C_(H)) and constant light(C_(L)) regions. As shown in FIG. 2, for an IgG antibody, the lightchain includes one variable region (V_(L)) and one constant region(C_(L)). An IgG heavy chain includes a variable region (V_(H)), a firstconstant region (C_(H)1), a hinge region, a second constant region(C_(H)2), and a third constant region (C_(H)3). In IgE and IgMantibodies, the heavy chain includes an additional constant region(C_(H)4).

Antibodies described herein can include, for example, monoclonalantibodies, polyclonal antibodies, multispecific antibodies, humanantibodies, humanized antibodies, camelized antibodies, chimericantibodies, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv), andanti-idiotypic (anti-Id) antibodies, and antigen-binding fragments ofany of the above. Antibodies can be of any type (e.g., IgG, IgE, IgM,IgD, IgA, or IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2)or subclass.

The term “Fc fragment,” as used herein, refers to one or more fragmentsof an Fc region that retains an Fc function and/or activity describedherein, such as binding to an Fc receptor. Examples of such fragmentsinclude fragments that include an N-linked glycosylation site of an Fcregion (e.g., an Asn297 of an IgG heavy chain or homologous sites ofother antibody isotypes), such as a CH2 domain. The term “antigenbinding fragment” of an antibody, as used herein, refers to one or morefragments of an antibody that retain the ability to specifically bind toan antigen. Examples of binding fragments encompassed within the term“antigen binding fragment” of an antibody include a Fab fragment, aF(ab′)2 fragment, a Fd fragment, a Fv fragment, a scFv fragment, a dAbfragment (Ward et al., (1989) Nature 341:544-546), and an isolatedcomplementarily determining region (CDR). These antibody fragments canbe obtained using conventional techniques known to those with skill inthe art, and the fragments can be screened for utility in the samemanner as are intact antibodies.

Reference Fc region-containing polypeptides described herein can beproduced by any method known in the art for the synthesis of antibodies(see, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988); Brinkman et al., 1995, J.Immunol. Methods 182:41-50; WO 92/22324; WO 98/46645).

Additional reference Fc region-containing polypeptides described hereinare bispecific antibodies and multivalent antibodies, as described in,e.g., Segal et al., J. Immunol. Methods 248:1-6 (2001); and Tutt et al.,J. Immunol. 147: 60 (1991).

C. Polypeptide Conjugates

The disclosure includes polypeptides (or Fc regions or Fc fragmentsthereof containing one or more N-glycosylation sites) that areconjugated or fused to one or more heterologous moieties and that havedifferent levels of sialylated glycans relative to a correspondingreference polypeptide. Heterologous moieties include, but are notlimited to, peptides, polypeptides, proteins, fusion proteins, nucleicacid molecules, small molecules, mimetic agents, synthetic drugs,inorganic molecules, and organic molecules. In some instances, areference polypeptide is a fusion protein that comprises a peptide,polypeptide, protein scaffold, scFv, dsFv, diabody, Tandab, or anantibody mimetic fused to an Fc region, such as a glycosylated Fcregion. The fusion protein can include a linker region connecting the Fcregion to the heterologous moiety (see, e.g., Hallewell et al. (1989),J. Biol. Chem. 264, 5260-5268; Alfthan et al. (1995), Protein Eng. 8,725-731; Robinson & Sauer (1996)).

In some instances, a reference fusion protein includes an Fc region (oran Fc fragment containing one or more N-glycosylation sites thereof)conjugated to a heterologous polypeptide of at least 10, at least 20, atleast 30, at least 40, at least 50, at least 60, at least 70, at least80, at least 90, or at least 100 amino acids.

In some instances, a reference fusion protein can include an Fc region(or Fc fragment containing one or more N-glycosylation sites thereof)conjugated to marker sequences, such as a peptide to facilitatepurification. A particular marker amino acid sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311). Otherpeptide tags useful for purification include, but are not limited to,the hemagglutinin “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767)and the “Flag” tag.

In other instances, a reference polypeptide (or an Fc region or Fcfragment containing one or more N-glycosylation sites thereof) isconjugated to a diagnostic or detectable agent. Such fusion proteins canbe useful for monitoring or prognosing the development or progression ofdisease or disorder as part of a clinical testing procedure, such asdetermining the efficacy of a particular therapy. Such diagnosis anddetection can be accomplished by coupling the polypeptide to detectablesubstances including, but not limited to, various enzymes, such as butnot limited to horseradish peroxidase, alkaline phosphatase,beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as,but not limited to, streptavidin/biotin and avidin/biotin; fluorescentmaterials, such as, but not limited to, umbelliferone, fluorescein,fluorescein isothiocynate, rhodamine, dichlorotriazinylaminefluorescein, dansyl chloride or phycoerythrin; luminescent materials,such as, but not limited to, luminol; bioluminescent materials, such asbut not limited to, luciferase, luciferin, and aequorin; radioactivematerials, such as but not limited to iodine (¹³¹I, ¹²⁵I, ¹²³I), carbon(¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹In),technetium (⁹⁹Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium(¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu,¹⁵³Gd, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷sc, ¹⁸⁶Re,¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ⁵¹Cr, ⁵⁴Mn,⁷⁵Se, ¹¹³Sn, and ¹¹⁷Sn; positron emitting metals using various positronemission tomographies, non-radioactive paramagnetic metal ions, andmolecules that are radiolabelled or conjugated to specificradioisotopes.

Techniques for conjugating therapeutic moieties to antibodies are wellknown (see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56. (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987)).

D. Sialyltransferase Polypeptides

Methods and compositions described herein include the use of asialyltransferase enzyme, e.g., an α 2,6 sialyltransferase (e.g., ST6Gal-I). A number of ST6 sialyltransferases are known in the art and arecommercially available (see, e.g., Takashima, Biosci. Biotechnol.Biochem. 72:1155-1167 (2008); Weinstein et al., J. Biol. Chem.262:17735-17743 (1987)). ST6 Gal-I catalyzes the transfer of sialic acidfrom a sialic acid donor (e.g., cytidine 5′-monophospho-N-acetylneuraminic acid) to a terminal galactose residue of glycans through an α2,6 linkage. The sialic acid donor reaction product is cytidine5′-monophosphate. FIGS. 3A-3C depict three exemplary ST6sialyltransferase amino acid sequences (SEQ ID NOs:1-3). In someembodiments, an ST6 sialyltransferase has or includes an amino acidsequence set forth in SEQ ID NO:1, SEQ ID NO:2, or in amino acidresidues 95-416 of SEQ ID NO:3, or a characteristic sequence elementthereof or therein. In some embodiments, an ST6 sialyltransferase has atleast 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%,87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%,73%, 72%, 71%, or 70% overall sequence identity with one or more of SEQID NO:1, SEQ ID NO:2, or amino acid residues 95-416 of SEQ ID NO:3.Alternatively or additionally, in some embodiments, an ST6sialyltransferase includes at least about 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 75, 100, or 150 or more contiguous amino acid residues found inSEQ ID NO:1, SEQ ID NO:2, or amino acid residues 95-416 of SEQ ID NO:3.

In some embodiments, an ST6 sialyltransferase differs from an amino acidsequence as set forth in SEQ ID NO:1, SEQ ID NO:2, or in amino acidresidues 95-416 of SEQ ID NO:3, or characteristic sequence elementsthereof or therein, by one or more amino acid residues. For example, insome embodiments, the difference is a conservative or nonconservativesubstitution of one or more amino acid residues. Conservativesubstitutions are those that substitute a given amino acid in apolypeptide by another amino acid of similar characteristics. Typicalconservative substitutions are the following replacements: replacementof an aliphatic amino acid, such as alanine, valine, leucine, andisoleucine, with another aliphatic amino acid; replacement of a serinewith a threonine or vice versa; replacement of an acidic residue, suchas aspartic acid and glutamic acid, with another acidic residue;replacement of a residue bearing an amide group, such as asparagine andglutamine, with another residue bearing an amide group; exchange of abasic residue, such as lysine and arginine, with another basic residue;and replacement of an aromatic residue, such as phenylalanine andtyrosine, with another aromatic residue.

In some embodiments, an ST6 sialyltransferase polypeptide includes asubstituent group on one or more amino acid residues. Still other usefulpolypeptides are associated with (e.g., fused, linked, or coupled to)another moiety (e.g., a peptide or molecule). For example, an ST6sialyltransferase polypeptides can be fused, linked, or coupled to anamino acid sequence (e.g., a leader sequence, a secretory sequence, aproprotein sequence, a second polypeptide, or a sequence thatfacilitates purification, enrichment, or stabilization of thepolypeptide).

II. Methods for Producing Sialylated Polypeptides

The present disclosure relates to Fc region-containing polypeptidepreparations (e.g., IVIg, Fc, or IgG antibodies) having higher levels ofbranched glycans that are sialylated on an α 1,3 and 1,6 arm of thebranched glycans in the Fc region (e.g., with a NeuAc-α 2,6-Gal orNeuAc-α 2,3-Gal terminal linkage), relative to a corresponding referencepolypeptide preparation. The higher levels can be measured on anindividual Fc region (e.g., an increase in the number of branchedglycans that are sialylated on an α 1,3 arm of the branched glycans inthe Fc region), or the overall composition of a preparation ofpolypeptides can be different (e.g., a preparation of polypeptides canhave a higher number or a higher percentage of branched glycans that aresialylated on an α 1,3 arm and an α 1,6 arm of the branched glycans inthe Fc region) relative to a corresponding preparation of referencepolypeptides).

In exemplary methods, Fc molecules were obtained or produced fromvarious sources, glycan compositions were characterized, and activitieswere determined. The Fc molecules were tested for their ability toincrease reticulated platelets in immune-related thrombocytopeniamodels.

ST6 Gal-I sialyltransferase catalyzes the transfer of sialic acid from asialic acid donor (e.g., cytidine 5′-monophospho-N-acetyl neuraminicacid) to a terminal galactose residue of glycans through an α 2,6linkage. The present disclosure exploits the discovery that ST6sialyltransferase catalyzes the transfer of sialic acid to branchedglycans (e.g., Fc branched glycans) comprising an α 1,3 arm and an α 1,6arm in an ordered fashion. As shown in FIG. 4, ST6 sialyltransferasetransfers a sialic acid to an α 1,3 arm of a branched glycan, which canbe followed by transfer of a second sialic acid to an α 1,6 arm(yielding a disialylated branched glycan), and can further be followedby removal of sialic acid from an α 1,3 arm (yielding a branched glycanhaving a sialic acid on an α 1,6 arm). Accordingly, by controllingand/or modulating activity (e.g., kinetics) of ST6 sialyltransferase,polypeptides having particular sialylation patterns can be produced.

Any parameter generally known to affect enzyme kinetics can becontrolled and/or modulated to produce a polypeptide preparation havinga predetermined level of sialic acid on an α 1,3 arm of a branchedglycan, on an α 1,6 arm of a branched glycan, and/or on an α 1,3 arm andan α 1,6 arm of a branched glycan. For example, reaction time, ST6sialyltransferase concentration and/or specific activity, branchedglycan concentration, sialic acid donor concentration, sialic acid donorreaction product concentration, pH, buffer composition, and/ortemperature can be controlled and/or modulated to produce a polypeptidepreparation having a desired level of sialylation (e.g., α 1,3 armand/or α 1,6 arm sialylation).

In some embodiments, to preferentially sialylate an α1,3 arm of branchedglycans (e.g., having an α 1,3 arm and an α 1,6 arm), branched glycansare contacted in vitro with an ST6 sialyltransferase under limitedreaction conditions. Such limited reaction conditions are selected suchthat addition of a sialic acid to an α 1,3 arm is enhanced relative toaddition of a sialic acid to an α 1,6 arm (e.g., rate of transfer of asialic acid to an α 1,3 arm (“R_(a) ^(1,3)”) exceeds rate of transfer ofa sialic acid to an α 1,6 arm (“R_(a) ^(1,6)”). In some embodiments,limited reaction conditions are further selected such that removal of asialic acid from an α1,6 arm is enhanced relative to addition of asialic acid to an α 1,6 arm (e.g., rate of removal of a sialic acid froman α 1,6 arm (“R_(r) ^(1,6)”) exceeds rate of transfer of a sialic acidto an α 1,6 arm (“R_(a) ^(1,6)”). Limited reaction conditions caninclude, for example, reduced reaction time, reduced enzymeconcentration and/or activity, reduced amount of branched glycans,reduced level of sialic acid donor, and/or reduced temperature.

In some embodiments, to preferentially sialylate an α1,6 arm of branchedglycans (e.g., having an α 1,3 arm and an α 1,6 arm), branched glycanscan be contacted in vitro with an ST6 sialyltransferase under extendedreaction conditions. Such extended reaction conditions are selected suchthat addition of a sialic acid to an α 1,6 arm is enhanced relative toremoval of a sialic acid from an α 1,6 arm (e.g., rate of transfer of asialic acid to an α 1,6 arm (“R_(a) ^(1,6)”) exceeds rate of removal ofa sialic acid from an α 1,6 arm (“R_(r) ^(1,6)”)). In some embodiments,extended reaction conditions are further selected such that, afterinitial conditions that enhance addition of sialic acid to an α 1,3 arm,conditions are extended such that removal of a sialic acid from an α 1,3arm is eventually enhanced relative to addition of a sialic acid to an α1,3 arm (e.g., rate of removal of a sialic acid from an α 1,3 arm(“R_(r) ^(1,3)”) exceeds rate of transfer of a sialic acid to an α 1,3arm (“R_(a) ^(1,3)”)). Extended reaction conditions can include, forexample, increased reaction time, increased enzyme concentration and/oractivity, increased amount of branched glycans, increased level ofsialic acid donor, and/or increased temperature.

In some embodiments, to preferentially sialylate both an α 1,3 arm andan α 1,6 arm of branched glycans (e.g., having an α 1,3 arm and an α 1,6arm), branched glycans are contacted in vitro with an ST6sialyltransferase under intermediate reaction conditions. Suchintermediate reaction conditions are selected such that addition of asialic acid to an α 1,3 arm is enhanced relative to removal of a sialicacid from an α 1,3 arm (e.g., rate of transfer of a sialic acid to an α1,3 arm (“R_(a) ^(1,3)”) exceeds rate of removal of a sialic acid froman α 1,3 arm (“R_(r) ^(1,3)”). In some embodiments, intermediatereaction conditions are further selected such that addition of a sialicacid to an α 1,6 arm is enhanced relative to removal of a sialic acidfrom an α 1,6 arm (e.g., rate of addition of a sialic acid to an α 1,6arm (“R_(a) ^(1,6)”) exceeds rate of removal of a sialic acid from an α1,6 arm (“R_(r) ^(1,6)”). Intermediate reaction conditions can include,for example, intermediate reaction time, intermediate enzymeconcentration and/or activity, intermediate amount of branched glycans,intermediate level of sialic acid donor, and/or intermediatetemperature. In some embodiments, intermediate reaction conditionsfurther include supplementing the sialic acid donor at least once duringthe reaction. In some embodiments, intermediate reaction conditionsfurther include removing a sialic acid donor reaction product at leastonce during the reaction. In some embodiments, intermediate reactionconditions further include supplementing the sialic acid donor reactionproduct at least once during the reaction.

In some embodiments, a polypeptide, e.g., a glycosylated antibody, issialylated after the polypeptide is produced. For example, a polypeptidecan be recombinantly expressed in a host cell (as described herein) andpurified using standard methods. The purified polypeptide is thencontacted with an ST6 sialyltransferase (e.g., a recombinantly expressedand purified ST6 sialyltransferase) in the presence of reactionconditions as described herein. In certain embodiments, the conditionsinclude contacting the purified polypeptide with an ST6sialyltransferase in the presence of a sialic acid donor, e.g., cytidine5′-monophospho-N-acetyl neuraminic acid, manganese, and/or otherdivalent metal ions. In some embodiments, IVIg is used in a sialylationmethod described herein.

In some embodiments, chemoenzymatic sialylation is used to sialylatepolypeptides. Briefly, this method involves sialylation of a purifiedbranched glycan, followed by incorporation of the sialylated branchedglycan en bloc onto a polypeptide to produce a sialylated polypeptide.

A branched glycan can be synthesized de novo using standard techniquesor can be obtained from a polypeptide preparation (e.g., a recombinantpolypeptide, Fc, or IVIg) using an appropriate enzyme, such as anendoglycosidase (e.g., EndoH or EndoF). After sialylation of thebranched glycan, the sialylated branched glycan can be conjugated to apolypeptide using an appropriate enzyme, such as a transglycosidase, toproduce a sialylated polypeptide.

In one exemplary method, a purified branched N-glycan is obtained from apolypeptide (e.g., a polypeptide preparation, e.g., IVIg) using anendoglycosidase. The purified branched N-glycan is then chemicallyactivated on the reducing end to form a chemically active intermediate.The branched N-glycan is then further processed, trimmed, and/orglycosylated using appropriate known glycosidases. The branched glycanis then sialylated using an ST6 sialylation as described herein. Afterengineering, the desired branched N-glycan is transferred onto apolypeptide using a transglycosidase (such as a transglycosidase inwhich glycosidic activity has been attenuated using geneticallyengineering).

In some embodiments, a branched glycan used in methods described hereinis a galactosylated branched glycan (e.g., includes a terminal galactoseresidue). In some embodiments, a branched glycan is galactosylatedbefore being sialylated using a method described herein. In someembodiments, a branched glycan is first contacted with agalactosyltransferase (e.g., a beta-1,3-galactosyltransferase) andsubsequently contacted with an ST6 sialyltransferase as describedherein. In some embodiments, a galactosylated glycan is purified beforebeing contacted with an ST6 sialyltransferase. In some embodiments, agalactosylated glycan is not purified before being contacted with an ST6sialyltransferase. In some embodiments, a branched glycan is contactedwith a galactosyltransferase and an ST6 sialyltransferase in a singlestep.

In some embodiments, a host cell is genetically engineered to express apolypeptide described herein and one or more sialyltransferase enzymes,e.g., an ST6 sialyltransferase. In some embodiments, the host cell isgenetically engineered to further express a galactosyltransferase. Thegenetically engineered host cell can be cultured under conditionssufficient to produce a particular sialylated polypeptide. For example,to produce polypeptides preferentially sialylated on α1,3 arms ofbranched glycans, a host cell can be genetically engineered to express arelatively low level of ST6 sialyltransferase, whereas to producepolypeptides preferentially sialylated on α1,6 arms of branched glycans,a host cell can be genetically engineered to express a relatively highlevel of ST6 sialyltransferase. In some embodiments, to producepolypeptides preferentially sialylated on α1,3 arms of branched glycans,a genetically engineered host cell can be cultured in a relatively lowlevel of sialic acid donor, whereas to produce polypeptidespreferentially sialylated on α1,6 arms of branched glycans, agenetically engineered host cell can be cultured in a relatively highlevel of sialic acid donor.

Recombinant expression of a gene, such as a nucleic acid encoding areference polypeptide and/or a sialtransferase described herein, caninclude construction of an expression vector containing a polynucleotidethat encodes a reference polypeptide and/or a sialtransferase. Once apolynucleotide has been obtained, a vector for the production of thereference polypeptide can be produced by recombinant DNA technologyusing techniques known in the art. Known methods can be used toconstruct expression vectors containing polypeptide coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination.

An expression vector can be transferred to a host cell by conventionaltechniques, and the transfected cells can then cultured by conventionaltechniques to produce reference polypeptides.

A variety of host expression vector systems can be used (see, e.g., U.S.Pat. No. 5,807,715). Such host-expression systems can be used to producepolypeptides and, where desired, subsequently purified. Such hostexpression systems include microorganisms such as bacteria (e.g., E.coli and B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing polypeptidecoding sequences; yeast (e.g., Saccharomyces and Pichia) transformedwith recombinant yeast expression vectors containing polypeptide codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing polypeptide codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g. Tiplasmid) containing polypeptide coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, NS0, and 3T3 cells) harboringrecombinant expression constructs containing promoters derived from thegenome of mammalian cells (e.g., metallothionein promoter) or frommammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

For bacterial systems, a number of expression vectors can be used,including, but not limited to, the E. coli expression vector pUR278(Ruther et al., 1983, EMBO 12:1791); pIN vectors (Inouye & Inouye, 1985,Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol.Chem. 24:5503-5509); and the like. pGEX vectors can also be used toexpress foreign polypeptides as fusion proteins with glutathione5-transferase (GST).

For expression in mammalian host cells, viral-based expression systemscan be utilized (see, e.g., Logan & Shenk, 1984, Proc. Natl. Acad. Sci.USA 8 1:355-359). The efficiency of expression can be enhanced by theinclusion of appropriate transcription enhancer elements, transcriptionterminators, etc. (see, e.g., Bittner et al., 1987, Methods in Enzymol.153:516-544).

In addition, a host cell strain can be chosen that modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the polypeptide expressed. Such cellsinclude, for example, established mammalian cell lines and insect celllines, animal cells, fungal cells, and yeast cells. Mammalian host cellsinclude, but are not limited to, CHO, VERY, BHK, HeLa, COS, MDCK, 293,3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, NS0 (a murine myelomacell line that does not endogenously produce any immunoglobulin chains),CRL7O3O and HsS78Bst cells.

For long-term, high-yield production of recombinant proteins, host cellsare engineered to stably express a polypeptide. Host cells can betransformed with DNA controlled by appropriate expression controlelements known in the art, including promoter, enhancer, sequences,transcription terminators, polyadenylation sites, and selectablemarkers. Methods commonly known in the art of recombinant DNA technologycan be used to select a desired recombinant clone.

In some embodiments, a reference Fc region-containing polypeptide isrecombinantly produced in cells as described herein, purified, andcontacted with a sialtransferase enzyme in vitro to produce Fcregion-containing polypeptides containing higher levels of glycanshaving higher levels of sialic acid on the α 1,3 arms and α 1,6 arms ofthe branched glycans with a NeuAc-α 2,6-Gal terminal linkage, relativeto the reference polypeptide. In some embodiments, a purified referencepolypeptide is contacted with the sialtransferase in the presence ofCMP-sialic acid, manganese, and/or other divalent metal ions.

A reference Fc region-containing polypeptide can be purified by anymethod known in the art for purification, for example, by chromatography(e.g., ion exchange, affinity, and sizing column chromatography),centrifugation, differential solubility, or by any other standardtechnique for the purification of proteins. For example, a referenceantibody can be isolated and purified by appropriately selecting andcombining affinity columns such as Protein A column with chromatographycolumns, filtration, ultra filtration, salting-out and dialysisprocedures (see Antibodies: A Laboratory Manual, Ed Harlow, David Lane,Cold Spring Harbor Laboratory, 1988). Further, as described herein, areference polypeptide can be fused to heterologous polypeptide sequencesto facilitate purification.

In some embodiments, a polypeptide can be purified using a lectin columnby methods known in the art (see, e.g., WO 02/30954). For example, apreparation of polypeptides can be enriched for polypeptides containingglycans having sialic acids in α 2,6 linkage as described in, e.g.,WO2008/057634. Following enrichment of polypeptides containing glycanshaving sialic acids in α 2,6 linkage, the glycan composition of suchpolypeptides can be further characterized to identify polypeptideshaving sialic acids attached to the α 1,3 arm and α 1,6 arm of abranched glycan. Preparations of polypeptides containing a predeterminedlevel of glycans having sialic acids in α 2,6 linkage on the α 1,3 armand α 1,6 arm can be selected for use, e.g., for therapeutic use. Suchcompositions can have increased levels of anti-inflammatory activity.

In accordance with the present disclosure, there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are described inthe literature (see, e.g., Green & Sambrook, Molecular Cloning: ALaboratory Manual, Fourth Edition (2012) Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.; DNA Cloning: A Practical Approach,Volumes I and II (Glover and Hames, eds. 1995); OligonucleotideSynthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames& S. J. Higgins eds. (1985)); Transcription And Translation (B. D. Hames& S. J. Higgins, eds. (1984)); R. I. Freshney, Culture of Animal Cells:A Manual of Basic Technique and Specialized Application (2010);Immobilized Cells and Enzymes (IRL Press, (1986)); J. M. Guisan,Immobilization of Enzymes and Cells (2013); B. Perbal, A Practical GuideTo Molecular Cloning (1984); T. A. Brown, Essential Molecular Biology: APractical Approach Volume I (2000); T. A. Brown, Essential MolecularBiology: A Practical Approach Volume 11(2002); F. M. Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994).

Glycan compositions can be characterized using methods described in,e.g., Barb, Biochemistry 48:9705-9707 (2009); Anumula, J. Immunol.Methods 382:167-176 (2012); Gilar et al., Analytical Biochem. 417:80-88(2011).

Glycan Evaluation

Glycans of polypeptides can be evaluated using any methods known in theart. For example, sialylation of glycan compositions (e.g., level ofbranched glycans that are sialylated on an α1,3 arm and/or an α1,6 arm)can be characterized using methods described in, e.g., Barb,Biochemistry 48:9705-9707 (2009); Anumula, J. Immunol. Methods382:167-176 (2012); Gilar et al., Analytical Biochem. 417:80-88 (2011);Wuhrer et al., J. Chromatogr. B. 849:115-128 (2007). In someembodiments, in addition to evaluation of sialylation of glycans, one ormore parameters described in Table 1 are evaluated.

In some instances, glycan structure and composition as described hereinare analyzed, for example, by one or more, enzymatic, chromatographic,mass spectrometry (MS), chromatographic followed by MS, electrophoreticmethods, electrophoretic methods followed by MS, nuclear magneticresonance (NMR) methods, and combinations thereof. Exemplary enzymaticmethods include contacting a polypeptide preparation with one or moreenzymes under conditions and for a time sufficient to release one ormore glycan(s) (e.g., one or more exposed glycan(s)). In some instances,the one or more enzymes include(s) PNGase F. Exemplary chromatographicmethods include, but are not limited to, Strong Anion Exchangechromatography using Pulsed Amperometric Detection (SAX-PAD), liquidchromatography (LC), high performance liquid chromatography (HPLC),ultra performance liquid chromatography (UPLC), thin layerchromatography (TLC), amide column chromatography, and combinationsthereof. Exemplary mass spectrometry (MS) include, but are not limitedto, tandem MS, LC-MS, LC-MS/MS, matrix assisted laser desorptionionisation mass spectrometry (MALDI-MS), Fourier transform massspectrometry (FTMS), ion mobility separation with mass spectrometry(IMS-MS), electron transfer dissociation (ETD-MS), and combinationsthereof. Exemplary electrophoretic methods include, but are not limitedto, capillary electrophoresis (CE), CE-MS, gel electrophoresis, agarosegel electrophoresis, acrylamide gel electrophoresis, SDS-polyacrylamidegel electrophoresis (SDS-PAGE) followed by Western blotting usingantibodies that recognize specific glycan structures, and combinationsthereof. Exemplary nuclear magnetic resonance (NMR) include, but are notlimited to, one-dimensional NMR (1D-NMR), two-dimensional NMR (2D-NMR),correlation spectroscopy magnetic-angle spinning NMR (COSY-NMR), totalcorrelated spectroscopy NMR (TOCSY-NMR), heteronuclear single-quantumcoherence NMR (HSQC-NMR), heteronuclear multiple quantum coherence(HMQC-NMR), rotational nuclear overhauser effect spectroscopy NMR(ROESY-NMR), nuclear overhauser effect spectroscopy (NOESY-NMR), andcombinations thereof.

In some instances, techniques described herein may be combined with oneor more other technologies for the detection, analysis, and or isolationof glycans or polypeptides. For example, in certain instances, glycansare analyzed in accordance with the present disclosure using one or moreavailable methods (to give but a few examples, see Anumula, Anal.Biochem., 350(1):1, 2006; Klein et al., Anal. Biochem., 179:162, 1989;and/or Townsend, R. R. Carbohydrate Analysis” High Performance LiquidChromatography and Capillary Electrophoresis, Ed. Z. El Rassi, pp181-209, 1995; WO2008/128216; WO2008/128220; WO2008/128218;WO2008/130926; WO2008/128225; WO2008/130924; WO2008/128221;WO2008/128228; WO2008/128227; WO2008/128230; WO2008/128219;WO2008/128222; WO2010/071817; WO2010/071824; WO2010/085251;WO2011/069056; and WO2011/127322, each of which is incorporated hereinby reference in its entirety). For example, in some instances, glycansare characterized using one or more of chromatographic methods,electrophoretic methods, nuclear magnetic resonance methods, andcombinations thereof. In some instances, methods for evaluating one ormore target protein specific parameters, e.g., in a polypeptidepreparation, e.g., one or more of the parameters disclosed herein, canbe performed by one or more of following methods.

TABLE 1 Exemplary methods of evaluating parameters: Method(s) Relevantliterature Parameter C18 UPLC Mass Spec.* Chen and Flynn, Anal.Biochem., Glycan(s) 370: 147-161 (2007) (e.g., N-linked glycan, exposedN- Chen and Flynn, J. Am. Soc. Mass linked glycan, glycan detection,Spectrom., 20: 1821-1833 (2009) glycan identification, andcharacterization; site specific glycation; glycoform detection (e.g.,parameters 1-7); percent glycosylation; and/or aglycosyl) Peptide LC-MSDick et al., Biotechnol. Bioeng., C-terminal lysine(reducing/non-reducing) 100: 1132-1143 (2008) Yan et al., J. Chrom. A.,1164: 153-161 (2007) Chelius et al., Anal. Chem., 78: 2370- 2376 (2006)Miller et al., J. Pharm. Sci., 100: 2543- 2550 (2011) LC-MS(reducing/non- Dick et al., Biotechnol. Bioeng., reducing/alkylated)100: 1132-1143 (2008) Goetze et al., Glycobiol., 21: 949-959 (2011) Weakcation exchange Dick et al., Biotechnol. Bioeng., (WCX) chromatography100: 1132-1143 (2008) LC-MS (reducing/non- Dick et al., Biotechnol.Bioeng., N-terminal pyroglu reducing/alkylated) 100: 1132-1143 (2008)Goetze et al., Glycobiol., 21: 949-959 (2011) PeptideLC-MS Yan et al.,J. Chrom. A., 1164: 153-161 (reducing/non-reducing) (2007) Chelius etal., Anal. Chem., 78: 2370- 2376 (2006) Miller et al., J. Pharm. Sci.,100: 2543- 2550 (2011) Peptide LC-MS Yan et al., J. Chrom. A., 1164:153-161 Methionine oxidation (reducing/non-reducing) (2007); Xie et al.,mAbs, 2: 379-394 (2010) Peptide LC-MS Miller et al., J. Pharm. Sci.,100: 2543- Site specific glycation (reducing/non-reducing) 2550 (2011)Peptide LC-MS Wang et al., Anal. Chem., 83: 3133-3140 Free cysteine(reducing/non-reducing) (2011); Chumsae et al., Anal. Chem., 81: 6449-6457 (2009) Bioanalyzer Forrer et al., Anal. Biochem., 334: 81-88 Glycan(e.g., N-linked glycan, (reducing/non-reducing)* (2004) exposed N-linkedglycan) (including, for example, glycan detection, identification, andcharacterization; site specific glycation; glycoform detection; percentglycosylation; and/or aglycosyl) LC-MS (reducing/non- Dick et al.,Biotechnol. Bioeng., Glycan (e.g., N-linked glycan, reducing/alkylated)*100: 1132-1143 (2008) exposed N-linked glycan) * Methods include Goetzeet al., Glycobiol., 21: 949-959 (including, for example, glycan removal(e.g., enzymatic, (2011) detection, identification, and chemical, andphysical) Xie et al., mAbs, 2: 379-394 (2010) characterization; sitespecific of glycans glycation; glycoform detection; percentglycosylation; and/or aglycosyl) Bioanalyzer Forrer et al., Anal.Biochem., 334: 81-88 Light chain: Heavy chain (reducing/non-reducing)(2004) Peptide LC-MS Yan et al., J. Chrom. A., 1164: 153-161Non-glycosylation-related peptide (reducing/non-reducing) (2007)modifications (including, for Chelius et al., Anal. Chem., 78: 2370-example, sequence analysis and 2376 (2006) identification of sequencevariants; Miller et al., J. Pharm. Sci., 100: 2543- oxidation;succinimide; aspartic 2550 (2011) acid; and/or site-specific asparticacid) Weak cation exchange Dick et al., Biotechnol. Bioeng., Isoforms(including, for example, (WCX) chromatography 100: 1132-1143 (2008)charge variants (acidic variants and basic variants); and/or deamidatedvariants) Anion-exchange Ahn et al., J. Chrom. B, 878: 403-408Sialylated glycan chromatography (2010) Anion-exchange Ahn et al., J.Chrom. B, 878: 403-408 Sulfated glycan chromatography (2010)1,2-diamino-4,5- Hokke et al., FEBS Lett., 275: 9-14 Sialic acidmethylenedioxybenzene (1990) (DMB) labeling method LC-MS Johnson et al.,Anal. Biochem., 360: 75- C-terminal amidation 83 (2007) LC-MS Johnson etal., Anal. Biochem., 360: 75- N-terminal fragmentation 83 (2007)Circular dichroism Harn et al., Current Trends in Secondary structure(including, for spectroscopy Monoclonal Antibody Development andexample, alpha helix content Manufacturing, S. J. Shire et al., eds,and/or beta sheet content) 229-246 (2010) Intrinsic and/or ANS dye Harnet al., Current Trends in Tertiary structure (including, forfluorescence Monoclonal Antibody Development and example, extent ofprotein folding) Manufacturing, S. J. Shire et al., eds, 229-246 (2010)Hydrogen-deuterium Houde et al., Anal. Chem., 81: 2644- Tertiarystructure and dynamics exchange-MS 2651 (2009) (including, for example,accessibility of amide protons to solvent water) Size-exclusionCarpenter et al., J. Pharm. Sci., Extent of aggregation chromatography99: 2200-2208 (2010) Analytical Pekar and Sukumar, Anal. Biochem.,ultracentrifugation 367: 225-237 (2007)

References listed in Table 1 are hereby incorporated by reference intheir entirety or, in the alternative, to the extent that they pertainto one or more of the methods disclosed in Table 1. Other methods forevaluating one or more parameters are disclosed in the examples.

III. Treatment of Immune-Related Thrombocytopenia

The inventors have discovered that biological activity of Fc-containingmolecules is enhanced by sialylation of two branches of branchedglycans. Accordingly, Fc region-containing polypeptides described herein(e.g., Fc region-containing polypeptides containing glycans containingsialic acid on an α 1,3 arm and an α 1,6 arm of branched glycans with aNeuAc-α 2,6-Gal terminal linkage) have increased activity relative to areference polypeptide. Current treatments for immune-relatedthrombocytopenia include IVIg infusions, platelets transfusions, andtreatment with thrombopoietin or thrombopoietin receptor agonist, e.g.,romiplostim (NPLATE®, Amgen) and eltrombopag (PROMACTA®,GlaxoSmithKline).

IV. Pharmaceutical Compositions and Administration

A polypeptide of the present disclosure, e.g., an Fc region-containingpolypeptide comprising branched glycans that are sialylated on both an α1,3 arm and an α 1,6 arm of the branched glycan in the Fc region, e.g.,with a NeuAc-α 2,6-Gal terminal linkage, can be incorporated into apharmaceutical composition and can be useful in the treatment ofimmune-related thrombocytopenia. Such a pharmaceutical composition isuseful as an improved composition for the prevention and/or treatment ofdiseases relative to the corresponding reference polypeptide.Pharmaceutical compositions comprising a polypeptide can be formulatedby methods known to those skilled in the art. The pharmaceuticalcomposition can be administered parenterally in the form of aninjectable formulation comprising a sterile solution or suspension inwater or another pharmaceutically acceptable liquid. For example, thepharmaceutical composition can be formulated by suitably combining thesulfated polypeptide with pharmaceutically acceptable vehicles or media,such as sterile water and physiological saline, vegetable oil,emulsifier, suspension agent, surfactant, stabilizer, flavoringexcipient, diluent, vehicle, preservative, binder, followed by mixing ina unit dose form required for generally accepted pharmaceuticalpractices. The amount of active ingredient included in thepharmaceutical preparations is such that a suitable dose within thedesignated range is provided.

The sterile composition for injection can be formulated in accordancewith conventional pharmaceutical practices using distilled water forinjection as a vehicle. For example, physiological saline or an isotonicsolution containing glucose and other supplements such as D-sorbitol,D-mannose, D-mannitol, and sodium chloride may be used as an aqueoussolution for injection, optionally in combination with a suitablesolubilizing agent, for example, alcohol such as ethanol and polyalcoholsuch as propylene glycol or polyethylene glycol, and a nonionicsurfactant such as polysorbate 80™, HCO-50 and the like.

Non-limiting examples of oily liquid include sesame oil and soybean oil,and it may be combined with benzyl benzoate or benzyl alcohol as asolubilizing agent. Other items that may be included are a buffer suchas a phosphate buffer, or sodium acetate buffer, a soothing agent suchas procaine hydrochloride, a stabilizer such as benzyl alcohol orphenol, and an antioxidant. The formulated injection can be packaged ina suitable ampoule.

Route of administration can be parenteral, for example, administrationby injection, transnasal administration, transpulmonary administration,or transcutaneous administration. Administration can be systemic orlocal by intravenous injection, intramuscular injection, intraperitonealinjection, subcutaneous injection.

The term “subcutaneous administration” refers to introduction of a drugunder the skin of an animal or human patient (e.g., by subcutaneousinfusion or subcutaneous bolus), preferably within a pocket between theskin and underlying tissue, by relatively slow, sustained delivery froma drug receptacle. The pocket may be created by pinching or drawing theskin up and away from underlying tissue. In particular embodiments, anextracellular matrix degrading enzyme (e.g., a hyaluronidase or anyextracellular matrix degrading enzyme described herein) is administeredat each of the sites (e.g., prior to administration of the compositionand/or during the non-delivery period). In particular embodiments, theextracellular matrix degrading enzyme is co-infused with thecomposition.

Convenient sites for subcutaneous administration include the shoulder,upper arm, thigh, and abdomen. In particular embodiments of the abovemethods, the composition is administered into subcutis or fat at a depthbetween 2 mm and 10 mm below the dermis of the subject.

The term “subcutaneous infusion” refers to introduction of a drug underthe skin of an animal or human patient, preferably within a pocketbetween the skin and underlying tissue, by relatively slow, sustaineddelivery from a drug receptacle for a period of time including, but notlimited to, 30 minutes or less, or 90 minutes or less. Optionally, theinfusion may be made by subcutaneous implantation of a drug deliverypump implanted under the skin of the animal or human patient, whereinthe pump delivers a predetermined amount of drug for a predeterminedperiod of time, such as 30 minutes, 90 minutes, or a time periodspanning the length of the treatment regimen.

The term “subcutaneous bolus” refers to drug administration beneath theskin of an animal or human patient, where bolus drug delivery ispreferably less than approximately 15 minutes, more preferably less than5 minutes, and most preferably less than 60 seconds. Administration ispreferably within a pocket between the skin and underlying tissue, wherethe pocket is created, for example, by pinching or drawing the skin upand away from underlying tissue.

The term “extracellular matrix degrading enzyme” means an enzyme thatcan break down extracellular matrix at the site of infusion, resultingin improved tissue permeability for an composition infused at the site.Extracellular matrix degrading enzymes include enzymes catalyzing thehydrolysis of hyaluronic acid (hyaluronan), a glycosaminoglycan,chondroitin, or collagen, such as a hyaluronidase, glycosaminoglycanase,collagenase (e.g. cathepsin), serine proteases, thiol proteases, andmatrix metalloproteases, of which the human enzymes are preferred andthe recombinant human enzymes are most preferred. Examples of suchenzymes which can be used in the methods and compositions of theinvention are described in U.S. Pat. Nos. 4,258,134; 4,820,516; 7,871,607; 7,767,429; 7,829,081; 7,846,431; 7,871,607; 8,187,855; and8,105,586, and U.S. Patent Publication Nos. 20090304665; 20110053247;20120101325; and 20110008309, each of which is incorporated byreference. Human hyaluronidases which can be used in the methods andcompositions of the invention are also described, for example, in U.S.Pat. Nos. 3,945,889; 6,057,110; 5,958,750; 5,854,046; 5,827,721; and5,747,027, each of which is incorporated herein by reference.Commercially available hyaluronidases which can be used in the methodsand compositions of the invention include HYDASE™ (PrimaPharm Inc.),VITRASSE® (ISTA Pharmaceuticals), AMPHADASE® (AmphastarPharmaceuticals), and HYLENEX® (sold by Halozyme Therapeutics).

A suitable means of administration can be selected based on the age andcondition of the patient. A single dose of the pharmaceuticalcomposition containing a modified polypeptide can be selected from arange of 0.001 to 1000 mg/kg of body weight. On the other hand, a dosecan be selected in the range of 0.001 to 100000 mg/body weight, but thepresent disclosure is not limited to such ranges. The dose and method ofadministration varies depending on the weight, age, condition, and thelike of the patient, and can be suitably selected as needed by thoseskilled in the art.

EXAMPLES Example 1—Preparation of Sialylated Glycoproteins

The sialylation of IVIg by the sialyltransferase ST6 was analyzed. IVIgwas first galactosylated and then sialylated. The reactions wereperformed sequentially. There was no purification betweengalactosylation and sialylation reactions. The relative abundance ofglycoforms was analyzed following the sialylation reactions.

Galactosylation

A reaction was set up that contained the following components at theconcentrations indicated in Table 2:

TABLE 2 Galactosylation conditions (Target s2IVIG) Final Constituentconcentration MOPS (pH 7.4) 50 mM MnCl₂ 8 mM IVIg 125 mg/ml B4GalT1 (100u/ml) 1.04 mg/g-IVIG UDP-Galactose 5 mM

The reaction was incubated for 24-72 hours at 37° C.

B. Sialylation

To an aliquot of the galactosylation reaction were added CMP-NANA, MOPSbuffer and ST6Gal1. The final volume was adjusted so that the finalconcentration of components in the reaction was as indicated in Table 3.

TABLE 3 Sialylation conditions Constituent Final concentration MOPS (pH7.4) 50 mM IVIg 115 mg/ml CMP-NANA (6 × 8 mM) 48 mM ST6Gal1 (SEQ IDNO: 1) 3.5 mg/g-IVIg

The reaction was incubated at 37° C. Aliquots were extracted at thetimes indicated in FIG. 5 and frozen at −20° C. for later analyses.

C. Results

As shown in FIG. 5, the predominant glycoform changed over time from G2Fto A1F (1,3) to A2F to A1F (1,6). The results are summarized in thereaction scheme depicted in FIG. 4. As shown in FIG. 4, the productglycoform can change between G2F, A1F (1,3), A2F, and A1F (1,6) duringthe course of a reaction due to competing addition (forward reaction)and removal (back reaction) steps.

The sialyltransferase ST6 can add sialic acid to either branch of asubstrate's biantennary N-glycan. However, these results demonstratethat addition to each branch happens at different rates, resulting indifferent end products depending on the reaction conditions. Addition ofsialic acid to the α1,3 branch is faster than addition to the α1,6branch.

These data also demonstrate that sialyltransferase ST6 can also catalyzethe removal of sialic acids from N-glycans. The removal of sialic acidfrom the α1,3 branch is faster than removal from the α1,6 branch. Thiscan surprisingly lead to the production of Fc glycans substantially orprimarily monosialylated on the α1,6 branch by modulating reactionconditions.

This Example demonstrates that reaction conditions can be controlled toproduce a glycoprotein product having a predetermined or targetsialylation levels. Such conditions can include time, ST6sialyltransferase concentration, substrate concentration, donor sugarnucleotide concentration, product nucleotide concentration, pH, buffercomposition, and/or temperature.

Example 2—Dose Response of IVIg, S1-IVIg, S2-IVIg, and Des-IVIg in aChronic ITP Mouse Model

The effect of IVIg, S1-IVIg, S2-IVIg, and Des-IVIg at varying doses inan anti-CD41 antibody mediated ITP mouse model was analyzed.

A. Study Design

Sixty-six to seventy two mice were given 1.5 μg/mouse of rat anti-CD41antibody (Ab) clone MWReg30 (BioLegend cat #133910) once daily for 4days (on Days 1, 2, 3 and 4), intraperitoneally. Six to twelve mice weredosed in the same manner with a rat IgG1, k isotype control (BioLegendcat #400414). All mice were dosed once intravenously with salinecontrol, IVIg, S1-IVIg, S2-IVIg, or desialylated-IVIg (Des-IVIg) atdifferent doses 1 to 2 hours after the third anti-CD41 Ab injection(Table 4). Mice were bled on Day 4 (4 h after the forth anti-CD41injection) and on Day 5 (24 h after the forth anti-CD41 Ab injection) toquantitate total platelet and reticulated platelet levels. To confirmthat platelet depletion was successful, a subgroup of mice was bled onDay 3, prior to treatment.

TABLE 4 IVIg, S1-IVIg, S2-IVIg, and Des-IVIg dose response study detailsInduction (1.5 μg IP) Treatment Agent Group # n 4 daily doses (200 uLIV) Dose Timing of Dosing Blood Sampling 1 6 anti-CD41 Saline 200 μL 1-2h post 3. anti-CD41 dose Day 3, 4 and Day 5 2 6 Rat IgG1 Saline 200 μL1-2 h post 3. anti-CD41 dose Day 4 and Day 5 3 8 anti-CD41 IVIgGammagard 0.5 g/kg 1-2 h post 3. anti-CD41 dose Day 4 and Day 5 4 8anti-CD41 IVIg Gammagard 1 g/kg 1-2 h post 3. anti-CD41 dose Day 4 andDay 5 5 8 anti-CD41 S1-IVIg 0.5 g/kg 1-2 h post 3. anti-CD41 dose Day 4and Day 5 6 8 anti-CD41 S1-IVIg 1 g/kg 1-2 h post 3. anti-CD41 dose Day4 and Day 5 7 8 anti-CD41 S2-IVIg 0.5 g/kg 1-2 h post 3. anti-CD41 doseDay 4 and Day 5 8 8 anti-CD41 S2-IVIg 1 g/kg 1-2 h post 3. anti-CD41dose Day 4 and Day 5 9 8 anti-CD41 Des-IVIg 0.5 g/kg 1-2 h post 3.anti-CD41 dose Day 4 and Day 5 10 8 anti-CD41 Des-IVIg 1 g/kg 1-2 h post3. anti-CD41 dose Day 4 and Day 5

B. Methods ITP Induction in Mice:

In vivo studies were conducted using female C57BL/6 mice (18-22 g,Charles Rivers Labs, MA). All procedures were performed in compliancewith the Animal Welfare Act and with the Guide for the Care and Use ofLaboratory Animals.

Quantitation of Total Platelets:

Blood samples were collected by submandibular bleed into EDTA coatedtubes, and then run on a VetScan Instrument for platelet leveldetermination. Total platelet levels were analyzed using One-Way ANOVAwith Dunnett's or Bonferroni's post-test.

Quantitation of Reticulated Platelets:

To evaluate and quantitate for the presence of reticulated (young)platelets which contain residual RNA, whole blood was sequentiallystained for total platelets (anti-CD61) followed by staining for the RNAwith thiazole orange (RNA-binding dye, commercially available asReticCount Reagent from BD Biosciences). This analysis was performed forblood samples collected on Day 5.

Ten microliters of whole blood was transferred into the bottom of a 5 mLFACS tube. Five microliters of anti-mouse CD61-PE antibody (BDBiosciences) was added directly to the whole blood and samples weremixed thoroughly by pipetting. Samples were incubated at roomtemperature for 5 minutes in the dark. Two milliliters of ReticCountreagent (BD Biosciences) was added to each sample and samples incubatedfor a minimum of 30 minutes at room temperature.

Samples were acquired on a FACS Canto flow cytometer (BD Biosciences).Total platelets were identified by forward and side scattercharacteristics of the cells and distinguished from erythrocytes bygating on CD61-PE positive events. A total 10,000 platelet events wererecorded for each sample. Using FlowJo software, a gate was set on thereticulated platelets (CD61 positive and thiazole orange positive) usingsamples from isotype control treated mice to achieve a rate of 6-10%reticulated platelets (normal rate). The same gate was then applied toall subsequent samples and treatment groups to calculate percentages ofreticulated and non-reticulated platelets. Total counts of reticulatedand non-reticulated platelets for each sample were calculated bymultiplying the total number of platelets measured in the VetScanInstrument by the percentage of the platelet fraction. Total reticulatedand non-reticulated platelet levels were analyzed using One-Way ANOVAwith Dunnett's or Bonferroni's post-test.

In addition to the overall platelet quantification using the VetScanInstrument, numbers of reticulated platelets were also determined usingReticCount, anti-CD61-PE labeled Ab and flow cytometry.

C. Results

The results of the IVIg, S1-IVIg, S2-IVIg, and Des-IVIg dose responsestudy are shown in Table 5.

TABLE 5 Total, reticulated, and non-reticulated platelet counts (10⁹/L)on Day 5 Isotype Disease Control Induction (1.5 μg) Anti-CD41 Ab (1.5μg) Treatment Saline Saline IVIg S1-IVIg S2-IVIg desialylated IVIg Dose0.5 g/kg 1 g/kg 0.5 g/kg 1 g/kg 0.5 g/kg 1 g/kg 0.5 g/kg 1 g/kg n pergroup 6 6 8 7 6 8 7 8 8 7 Total 733 ± 73 240 ± 200 282 ± 79  429 ± 116 355 ± 137 356 ± 89 362 ± 94  501 ± 102 224 ± 51 182 ± 78 Platelets ×10⁹/L [mean ± SD] Reticulated  70 ± 18 97 ± 24 109 ± 76 268 ± 84 196 ±64 234 ± 81 304 ± 73 391 ± 86 175 ± 42 134 ± 56 Platelets × 10⁹/L [mean± SD] Non-Reticulated 663 ± 64 143 ± 182 173 ± 49 161 ± 77 159 ± 85 122± 63  58 ± 41 111 ± 69  49 ± 32  48 ± 26 Platelets × 10⁹/L [mean ± SD]

Example 3—Comparison of IVIg, S1-IVIg, S2-IVIg, and Des-IVIg in aChronic ITP Mouse Model

The effect of IVIg, S1-IVIg, S2-IVIg, and Des-IVIg in an anti-CD41antibody mediated ITP mouse model was analyzed.

A. Study Design

Sixty-six to seventy two mice were given 1.5 μg/mouse of rat anti-CD41antibody (Ab) clone MWReg30 (BioLegend cat #133910) once daily for 4days (on Days 1, 2, 3 and 4), intraperitoneally. Six to twelve mice weredosed in the same manner with a rat IgG1, k isotype control (BioLegendcat #400414). All mice were dosed once intravenously with salinecontrol, IVIg, S1-IVIg, S2-IVIg, or desialylated-IVIg (Des-IVIg atdifferent doses 1 to 2 hours after the third anti-CD41 Ab injection(Table 6). Mice were bled on Day 4 (4 h after the forth anti-CD41injection) and on Day 5 (24 h after the forth anti-CD41 Ab injection) toquantitate total platelet and reticulated platelet levels. To confirmthat platelet depletion was successful, a subgroup of mice was bled onDay 3, prior to treatment. On Day 4 bone marrow cells were isolated toquantitate megakaryocytes.

TABLE 6 IVIg, S1-IVIg, S2-IVIg, and Des-IVIg comparison study detailsInduction (1.5 μg IP) Treatment Agent Group # n 4 daily doses (200 uLIV) Dose Timing of Dosing Blood Sampling 1 12 anti-CD41 Saline 200 μL1-2 h post 3. anti-CD41 dose Day 3, Day 4, and Day 5 2 12 Rat IgG1Saline 200 μL 1-2 h post 3. anti-CD41 dose Day 4 and Day 5 3 12anti-CD41 IVIg Gammagard 1 g/kg 1-2 h post 3. anti-CD41 dose Day 4 andDay 5 4 12 anti-CD41 S1-IVIg 1 g/kg 1-2 h post 3. anti-CD41 dose Day 4and Day 5 5 12 anti-CD41 S2-IVIg 1 g/kg 1-2 h post 3. anti-CD41 dose Day4 and Day 5 6 12 anti-CD41 Des-IVIg 1 g/kg 1-2 h post 3. anti-CD41 doseDay 4 and Day 5

B. Methods ITP Induction in Mice:

In vivo studies were conducted using female C57BL/6 mice (18-22 g,Charles Rivers Labs, MA). All procedures were performed in compliancewith the Animal Welfare Act and with the Guide for the Care and Use ofLaboratory Animals.

Quantitation of Total Platelets:

Blood samples were collected by submandibular bleed into EDTA coatedtubes, and then run on a VetScan Instrument for platelet leveldetermination. Total platelet levels were analyzed using One-Way ANOVAwith Dunnett's or Bonferroni's post-test.

Quantitation of Reticulated Platelets:

To evaluate and quantitate for the presence of reticulated (young)platelets which contain residual RNA, whole blood was sequentiallystained for total platelets (anti-CD61) followed by staining for the RNAwith thiazole orange (RNA-binding dye, commercially available asReticCount Reagent from BD Biosciences). This analysis was performed forblood samples collected on Day 5.

Ten microliters of whole blood was transferred into the bottom of a 5 mLFACS tube. Five microliters of anti-mouse CD61-PE antibody (BDBiosciences) was added directly to the whole blood and samples weremixed thoroughly by pipetting. Samples were incubated at roomtemperature for 5 minutes in the dark. Two milliliters of ReticCountreagent (BD Biosciences) was added to each sample and samples incubatedfor a minimum of 30 minutes at room temperature.

Samples were acquired on a FACS Canto flow cytometer (BD Biosciences).Total platelets were identified by forward and side scattercharacteristics of the cells and distinguished from erythrocytes bygating on CD61-PE positive events. A total 10,000 platelet events wererecorded for each sample. Using FlowJo software, a gate was set on thereticulated platelets (CD61 positive and thiazole orange positive) usingsamples from isotype control treated mice to achieve a rate of 6-10%reticulated platelets (normal rate). The same gate was then applied toall subsequent samples and treatment groups to calculate percentages ofreticulated and non-reticulated platelets. Total counts of reticulatedand non-reticulated platelets for each sample were calculated bymultiplying the total number of platelets measured in the VetScanInstrument by the percentage of the platelet fraction. Total reticulatedand non-reticulated platelet levels were analyzed using One-Way ANOVAwith Dunnett's or Bonferroni's post-test.

In addition to the overall platelet quantification using the VetScanInstrument, numbers of reticulated platelets were also determined usingReticCount, anti-CD61-PE labeled Ab and flow cytometry.

Quantitation of Megakaryocytes in the Bone Marrow:

On Day 4 (1 day after IVIg treatment and 4 h after the 4^(th) anti-CD41antibody injection) of the study, bone marrow was extracted from onefemur per mouse by using a syringe with a 25 gauge needle, flushing thebone shaft repeatedly with 0.5 mL of media. Cell suspensions werefiltered through a nylon mesh and fixed in 4% paraformaldehyde for 15minutes on ice. Cells were washed twice with PBS buffer containing 10%culture grade normal bovine serum, resuspended and counted using aViCell cell counter. Cells were resuspended at 1×10⁶ cells/mL. Cytospinslides were prepared with 0.5 mL per slide. The slides were air-driedand stored at 80° C. until use.

After blocking, cells were stained with anti-CD41 (rat anti-mouse CD41;clone: MWReg30, cat #133910, Biolegend; diluted 1:150 in PBS contains10% normal donkey serum) by immunohistochemistry using a BondMaxinstrument (Leica) and the Rat Polink-2 open kit protocol. Slides werecounter stained with hematoxylin, mounted, and cover slipped.

Stained slides were imaged using a Vectra microscope system under 4× and20× magnification. Images were spectrally unmixed, segmented, thenquantified for megakaryocyte count as well as CD41 signal intensityusing Inform software. Total, mean and maximum signals as well as signalarea was calculated for each category. Data were normalized to totalcell numbers and reported as total OD signal or per cell ratio. Datawere transferred into Excel and Graph Pad Prism, graphed and analyzedfor statistically significant differences.

C. Results

The results of the IVIg, S1-IVIg, S2-IVIg, and Des-IVIg comparison studyare shown in Table 7.

TABLE 7 Total, reticulated, and non-reticulated platelet counts (10⁹/L)on Day 5 and Megakaryocyte count in bone marrow cells (MK/10⁶ BM cells)on Day 4 Isotype Control Disease Induction (1.5 μg) Anti-CD41 Ab (1.5μg) Treatment Saline Saline IVIg S1-IVIg S2-IVIg Desialylated IVIg Dose1 g/kg 1 g/kg 1 g/kg 1 g/kg n per group 6 6 6 6 6 6 Total Platelets 739± 84  277 ± 203 290 ± 113 563 ± 138 578 ± 209 220 ± 97 [mean ± SD]Reticulated 61 ± 11 98 ± 57 131 ± 60  184 ± 34  261 ± 49  130 ± 46Platelets [mean ± SD] Non-Reticulated 679 ± 75  179 ± 168 159 ± 60  379± 133 318 ± 177  90 ± 56 Platelets [mean ± SD] Megakaryocytes 328 ± 132323 ± 51  341 ± 162 360 ± 31  496 ± 115 372 ± 87 in Bone Marrow [mean ±SD]

Example 4—Comparison of IVIg, rFc, S1-rFc, S2-rFc, and Des-IVIg in aChronic ITP Mouse Model

The effect of IVIg, rFc, S1-rFc, S2-rFc, and Des-IVIg in an anti-CD41antibody mediated ITP mouse model was analyzed.

A. Study Design

Sixty-six to seventy two mice were given 1.5 μg/mouse of rat anti-CD41antibody (Ab) clone MWReg30 (BioLegend cat #133910) once daily for 4days (on Days 1, 2, 3 and 4), intraperitoneally. Six to twelve mice weredosed in the same manner with a rat IgG1, k isotype control (BioLegendcat #400414). All mice were dosed once intravenously with salinecontrol, IVIg, recombinant Fc (rFc), S1-rFc, S2-rFc, or Des-IVIg atdifferent doses 1 to 2 hours after the third anti-CD41 Ab injection(Table 8). Mice were bled on Day 4 (4 h after the forth anti-CD41injection) and on Day 5 (24 h after the forth anti-CD41 Ab injection) toquantitate total platelet and reticulated platelet levels. To confirmthat platelet depletion was successful, a subgroup of mice was bled onDay 3, prior to treatment. On Day 5 bone marrow cells were isolated toquantitate megakaryocytes.

TABLE 8 IVIg, rFc, S1-rFc, S2-rFc, and Des-IVIg comparison study detailsInduction (1.5 μg IP) Treatment Agent Group # n 4 daily doses (200 uLIV) Dose Timing of Dosing Blood Sampling 1 12 anti-CD41 Saline 200 μL1-2 h post 3. anti-CD41 dose Day 3, Day 4, and Day 5 2 12 Rat IgG1Saline 200 μL 1-2 h post 3. anti-CD41 dose Day 4 and Day 5 3 12anti-CD41 IVIg Gammagard 1 g/kg 1-2 h post 3. anti-CD41 dose Day 4 andDay 5 4 12 anti-CD41 rFc 0.3 g/kg 1-2 h post 3. anti-CD41 dose Day 4 andDay 5 5 12 anti-CD41 S1-rFc 0.3 g/kg 1-2 h post 3. anti-CD41 dose Day 4and Day 5 6 17 anti-CD41 S2-rFc 0.3 g/kg 1-2 h post 3. anti-CD41 doseDay 4 and Day 5 7 12 anti-CD41 Des-IVIg 1 g/kg 1-2 h post 3. anti-CD41dose Day 4 and Day 5

B. Methods ITP Induction in Mice:

In vivo studies were conducted using female C57BL/6 mice (18-22 g,Charles Rivers Labs, MA). All procedures were performed in compliancewith the Animal Welfare Act and with the Guide for the Care and Use ofLaboratory Animals.

Quantitation of Total Platelets:

Blood samples were collected by submandibular bleed into EDTA coatedtubes, and then run on a VetScan Instrument for platelet leveldetermination. Total platelet levels were analyzed using One-Way ANOVAwith Dunnett's or Bonferroni's post-test.

Quantitation of Reticulated Platelets:

To evaluate and quantitate for the presence of reticulated (young)platelets which contain residual RNA, whole blood was sequentiallystained for total platelets (anti-CD61) followed by staining for the RNAwith thiazole orange (RNA-binding dye, commercially available asReticCount Reagent from BD Biosciences). This analysis was performed forblood samples collected on Day 5.

Ten microliters of whole blood was transferred into the bottom of a 5 mLFACS tube. Five microliters of anti-mouse CD61-PE antibody (BDBiosciences) was added directly to the whole blood and samples weremixed thoroughly by pipetting. Samples were incubated at roomtemperature for 5 minutes in the dark. Two milliliters of ReticCountreagent (BD Biosciences) was added to each sample and samples incubatedfor a minimum of 30 minutes at room temperature.

Samples were acquired on a FACS Canto flow cytometer (BD Biosciences).Total platelets were identified by forward and side scattercharacteristics of the cells and distinguished from erythrocytes bygating on CD61-PE positive events. A total 10,000 platelet events wererecorded for each sample. Using FlowJo software, a gate was set on thereticulated platelets (CD61 positive and thiazole orange positive) usingsamples from isotype control treated mice to achieve a rate of 6-10%reticulated platelets (normal rate). The same gate was then applied toall subsequent samples and treatment groups to calculate percentages ofreticulated and non-reticulated platelets. Total counts of reticulatedand non-reticulated platelets for each sample were calculated bymultiplying the total number of platelets measured in the VetScanInstrument by the percentage of the platelet fraction. Total reticulatedand non-reticulated platelet levels were analyzed using One-Way ANOVAwith Dunnett's or Bonferroni's post-test.

In addition to the overall platelet quantification using the VetScanInstrument, numbers of reticulated platelets were also determined usingReticCount, anti-CD61-PE labeled Ab and flow cytometry.

Quantitation of Megakaryocytes in the Bone Marrow:

On Day 5 of the study (ITP-010; 2 days after IVIg treatment and 24 hafter the 4^(th) anti-CD41 antibody injection), bone marrow wasextracted from one femur per mouse by using a syringe with a 25 gaugeneedle, flushing the bone shaft repeatedly with 0.5 mL of media. Cellsuspensions were filtered through a nylon mesh and fixed in 4%paraformaldehyde for 15 minutes on ice. Cells were washed twice with PBSbuffer containing 10% culture grade normal bovine serum, resuspended andcounted using a ViCell cell counter. Cells were resuspended at 1×10⁶cells/mL. Cytospin slides were prepared with 0.5 mL per slide. Theslides were air-dried and stored at 80° C. until use.

After blocking, cells were stained with anti-CD41 (rat anti-mouse CD41;clone: MWReg30, cat #133910, Biolegend; diluted 1:150 in PBS contains10% normal donkey serum) by immunohistochemistry using a BondMaxinstrument (Leica) and the Rat Polink-2 open kit protocol. Slides werecounter stained with hematoxylin, mounted, and cover slipped.

Stained slides were imaged using a Vectra microscope system under 4x and20× magnification. Images were spectrally unmixed, segmented, thenquantified for megakaryocyte count as well as CD41 signal intensityusing Inform software. Total, mean and maximum signals as well as signalarea was calculated for each category. Data were normalized to totalcell numbers and reported as total OD signal or per cell ratio. Datawere transferred into Excel and Graph Pad Prism, graphed and analyzedfor statistically significant differences.

C. Results

The results of IVIg, rFc, S1-rFc, S2-rFc, and Des-IVIg comparison studyare shown in Table 9.

TABLE 9 Total, reticulated, and non-reticulated platelet counts (10⁹/L)and Megakaryocyte count in bone marrow cells (MK/10⁶ BM cells) on Day 5Isotype Control Disease Induction (1.5 μg) Anti-CD41 Ab (1.5 μg)Treatment Saline Saline IVIg rFc S1-rFc S2-rFc Des IVIg Dose 1 g/kg 0.3g/kg 0.3 g/kg 0.3 g/kg 1 g/kg n per group 6 6 6 6 6 6 6 Total Platelets734 ± 124 189 ± 83  401 ± 103 277 ± 226 379 ± 198 369 ± 89 160 ± 67[mean ± SD] Reticulated 64 ± 10 73 ± 39 125 ± 42  116 ± 55  178 ± 60 234 ± 95 122 ± 56 platelets [mean ± SD] Non-Reticulated 670 ± 117 116 ±70  277 ± 106 161 ± 191 201 ± 157 135 ± 46  38 ± 25 platelets [mean ±SD] Megakaryocytes in 126 ± 64  1.0 ± 0.7 275 ± 87  237 ± 57  218 ± 68 345 ± 44  81 ± 53 Bone Marrow [mean ± SD]

While the methods have been described in conjunction with variousinstances and examples, it is not intended that the methods be limitedto such instances or examples. On the contrary, the methods encompassvarious alternatives, modifications, and equivalents, as will beappreciated by those of skill in the art.

What is claimed is:
 1. A pharmaceutical preparation comprisingsialylated IVIG, wherein at least 65% of branched glycans are sialylatedon both the on both the α 1,3 arm and the α 1,6 arm by way of NeuAc-α2,6-Gal terminal linkages; at least 60% of the branched glycans on theFab domain are sialylated on both the on both the α 1,3 arm and the α1,6 arm by way of NeuAc-α 2,6-Gal terminal linkages; and at least 80% ofthe branched glycan on the Fc domain are sialylated on both the on boththe α 1,3 arm and the α 1,6 arm by way of NeuAc-α 2,6-Gal terminallinkages.